Inbred corn plant 29MIFI2 and seeds thereof

ABSTRACT

According to the invention, there is provided an inbred corn plant designated 29MIF12. This invention thus relates to the plants, seeds and tissue cultures of the inbred corn plant 29MIF12, and to methods for producing a corn plant produced by crossing the inbred plant 29MIF12 with itself or with another corn plant, such as another inbred. This invention further relates to corn seeds and plants produced by crossing the inbred plant 29MIF12 with another corn plant, such as another inbred, and to crosses with related species. This invention further relates to the inbred and hybrid genetic complements of the inbred corn plant 29MIF12, and also to the RFLP and genetic isozyme typing profiles of inbred corn plant 29MIF12.

BACKGROUND OF THE INVENTION

I. Technical Field of the Invention

The present invention relates to the field of corn breeding. Inparticular, the invention relates to the inbred corn seed and plantdesignated 29MIFI2, and derivatives and tissue cultures of such inbredplant.

II. Description of the Background Art

The goal of field crop breeding is to combine various desirable traitsin a single variety/hybrid. Such desirable traits include greater yield,better stalks, better roots, resistance to insecticides, herbicides,pests, and disease, tolerance to heat and drought, reduced time to cropmaturity, better agronomic quality, and uniformity in germination times,stand establishment, growth rate, maturity, and fruit size.

Breeding techniques take advantage of a plant's method of pollination.There are two general methods of pollination: a plant self-pollinates ifpollen from one flower is transferred to the same or another flower ofthe same plant. A plant cross-pollinates if pollen comes to it from aflower on a different plant.

Corn plants (Zea mays L.) can be bred by both self-pollination andcross-pollination. Both types of pollination involve the corn plant'sflowers. Corn has separate male and female flowers on the same plant,located on the tassel and the ear, respectively. Natural pollinationoccurs in corn when wind blows pollen from the tassels to the silks thatprotrude from the tops of the ear shoot.

Plants that have been self-pollinated and selected for type over manygenerations become homozygous at almost all gene loci and produce auniform population of true breeding progeny, a homozygous plant. A crossbetween two such homozygous plants produce a uniform population ofhybrid plants that are heterozygous for many gene loci. Conversely, across of two plants each heterozygous at a number of gene loci producesa population of hybrid plants that differ genetically and are notuniform. The resulting non-uniformity makes performance unpredictable.

The development of uniform corn plant hybrids requires the developmentof homozygous inbred plants, the crossing of these inbred plants, andthe evaluation of the crosses. Pedigree breeding and recurrent selectionare examples of breeding methods used to develop inbred plants frombreeding populations. Those breeding methods combine the geneticbackgrounds from two or more inbred plants or various other broad-basedsources into breeding pools from which new inbred plants are developedby selfing and selection of desired phenotypes. The new inbreds arecrossed with other inbred plants and the hybrids from these crosses areevaluated to determine which of those have commercial potential.

The pedigree breeding method for single-gene traits involves crossingtwo genotypes. Each genotype can have one or more desirablecharacteristics lacking in the other; or, each genotype can complementthe other. If the two original parental genotypes do not provide all ofthe desired characteristics, other genotypes can be included in thebreeding population. Superior plants that are the products of thesecrosses are selfed and selected in successive generations. Eachsucceeding generation becomes more homogeneous as a result ofself-pollination and selection. Typically, this method of breedinginvolves five or more generations of selfing and selection: S₁→S₂;S₂→S₃; S₃→S₄; S₄→S₅, etc. After at least five generations, the inbredplant is considered genetically pure.

Backcrossing can also be used to improve an inbred plant. Backcrossingtransfers a specific desirable trait from one inbred or other source toan inbred that lacks that trait. This can be accomplished for example byfirst crossing a superior inbred (A) (recurrent parent) to a donorinbred (non-recurrent parent), which carries the appropriate gene(s) forthe trait in question. The progeny of this cross are then mated back tothe superior recurrent parent (A) followed by selection in the resultantprogeny for the desired trait to be transferred from the non-recurrentparent. After five or more backcross generations with selection for thedesired trait, the progeny are heterozygous for loci controlling thecharacteristic being transferred, but are like the superior parent formost or almost all other genes. The last backcross generation would beselfed to give pure breeding progeny for the gene(s) being transferred.

A single cross hybrid corn variety is the cross of two inbred plants,each of which has a genotype which complements the genotype of theother. The hybrid progeny of the first generation is designated F₁.Preferred F₁ hybrids are more vigorous than their inbred parents. Thishybrid vigor, or heterosis, is manifested in many polygenic traits,including markedly improved higher yields, better stalks, better roots,better uniformity and better insect and disease resistance. In thedevelopment of hybrids only the F₁ hybrid plants are sought. An F₁single cross hybrid is produced when two inbred plants are crossed. Adouble cross hybrid is produced from four inbred plants crossed in pairs(A×B and C×D) and then the two F₁ hybrids are crossed again (A×B)×(C×D).

The development of a hybrid corn variety involves three steps: (1) theselection of plants from various germplasm pools; (2) the selfing of theselected plants for several generations to produce a series of inbredplants, which, although different from each other, each breed true andare highly uniform; and (3) crossing the selected inbred plants withunrelated inbred plants to produce the hybrid progeny (F₁). During theinbreeding process in corn, the vigor of the plants decreases. Vigor isrestored when two unrelated inbred plants are crossed to produce thehybrid progeny (F₁). An important consequence of the homozygosity andhomogeneity of the inbred plants is that the hybrid between any twoinbreds is always the same. Once the inbreds that give a superior hybridhave been identified, hybrid seed can be reproduced indefinitely as longas the homogeneity of the inbred parents is maintained. Conversely, muchof the hybrid vigor exhibited by F₁ hybrids is lost in the nextgeneration (F₂). Consequently, seed from hybrid varieties is not usedfor planting stock. It is not generally beneficial for farmers to saveseed of F₁ hybrids. Rather, farmers purchase F₁ hybrid seed for plantingevery year.

North American farmers plant over 70 million acres of corn at thepresent time and there are extensive national and internationalcommercial corn breeding programs. A continuing goal of these cornbreeding programs is to develop high-yielding corn hybrids that arebased on stable inbred plants that maximize the amount of grain producedand minimize susceptibility to environmental stresses. To accomplishthis goal, the corn breeder must select and develop superior inbredparental plants for producing hybrids.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides a corn plant designated29MIFI2. Also provided are corn plants having all the physiological andmorphological characteristics of corn plant 29MIFI2.

The inbred corn plant of the invention may further comprise, or have, acytoplasmic factor that is capable of conferring male sterility. Partsof the corn plant of the present invention are also provided, such as,e.g., pollen obtained from an inbred plant and an ovule of the inbredplant.

The invention also concerns seed of the corn plant 29MIFI2, which hasbeen deposited with the ATCC. The invention thus provides inbred cornseed designated 29MIFI2, and having ATCC Accession No. 209896.

The inbred corn seed of the invention may be provided as an essentiallyhomogeneous population of inbred corn seed designated 29MIFI2.

Essentially homogeneous populations of inbred seed are those thatconsist essentially of the particular inbred seed, and are generallypurified free from substantial numbers of other seed, so that the inbredseed forms between about 90% and about 100% of the total seed, andpreferably, between about 95% and about 100% of the total seed. Mostpreferably, an essentially homogeneous population of inbred corn seedwill contain between about 98.5%, 99%, 99.5% and about 100% of inbredseed, as measured by seed grow outs.

In any event, even if a population of inbred corn seed was found, forsome reason, to contain about 50%, or even about 20% or 15% of inbredseed, this would still be distinguished from the small fraction ofinbred seed that may be found within a population of hybrid seed, e.g.,within a bag of hybrid seed. In such a bag of hybrid seed offered forsale, the Governmental regulations require that the hybrid seed be atleast about 95% of the total seed. In the practice of the presentinvention, the hybrid seed generally forms at least about 97% of thetotal seed. In the most preferred practice of the invention, the femaleinbred seed that may be found within a bag of hybrid seed will be about1% of the total seed, or less, and the male inbred seed that may befound within a bag of hybrid seed will be negligible, i.e., will be onthe order of about a maximum of 1 per 100,000, and usually less thanthis value.

The population of inbred corn seed of the invention is furtherparticularly defined as being essentially free from hybrid seed. Theinbred seed population may be separately grown to provide an essentiallyhomogeneous population of inbred corn plant designated 29MIFI2.

In another aspect, the present invention provides for single geneconverted plants of 29MIFI2. The single transferred gene may preferablybe a dominant or recessive allele. Preferably the single transferredgene will confer such traits as male sterility, herbicide resistance,insect resistance, resistance for bacterial, fungal, or viral disease,male fertility, enhanced nutritional quality, and industrial usage. Thesingle gene may be a naturally occurring maize gene or a transgeneintroduced through genetic engineering techniques.

In another aspect, the present invention provides a tissue culture ofregenerable cells of inbred corn plant 29MIFI2. The tissue culture willpreferably be capable of regenerating plants having the physiologicaland morphological characteristics of the foregoing inbred corn plant,and of regenerating plants having substantially the same genotype as theforegoing inbred corn plant. Preferably, the regenerable cells in suchtissue cultures will be embryos, protoplasts, meristematic cells,callus, pollen, leaves, anthers, roots, root tips, silk, flowers,kernels, ears, cobs, husks or stalks. Still further, the presentinvention provides corn plants regenerated from the tissue cultures ofthe invention.

In yet another aspect, the present invention provides processes forpreparing corn seed or plants, which processes generally comprisecrossing a first parent corn plant with a second parent corn plant,wherein at least one of the first or second parent corn plants is theinbred corn plant designated 29MIFI2. These processes may be furtherexemplified as processes for preparing hybrid corn seed or plants,wherein a first inbred corn plant is crossed with a second, distinctinbred corn plant to provide a hybrid that has, as one of its parents,the inbred corn plant 29MIFI2.

In a preferred embodiment, crossing comprises planting, in pollinatingproximity, seeds of the first and second parent corn plant, andpreferably, seeds of a first inbred corn plant and a second, distinctinbred corn plant; cultivating or growing the seeds of said first andsecond parent corn plants into plants that bear flowers; emasculatingthe male flowers of the first or second parent corn plant, i.e.,treating the flowers so as to prevent pollen production, in order toproduce an emasculated parent corn plant; allowing naturalcross-pollination to occur between the first and second parent cornplants; and harvesting the seeds from the emasculated parent corn plant.Where desired, the harvested seed is grown to produce a corn plant orhybrid corn plant.

The present invention also provides corn seed and plants produced by aprocess that comprises crossing a first parent corn plant with a secondparent corn plant, wherein at least one of the first or second parentcorn plants is the inbred corn plant designated 29MIFI2. In oneembodiment, corn plants produced by the process are first generation(F₁) hybrid corn plants produced by crossing an inbred in accordancewith the invention with another, distinct inbred. The present inventionfurther contemplates seed of an F₁ hybrid corn plant.

In certain exemplary embodiments, the invention provides an F₁ hybridcorn plant and seed thereof, which hybrid corn plant is designated1131838, having 29MIFI2 as one inbred parent.

In yet a further aspect, the invention provides an inbred geneticcomplement of the corn plant designated 29MIFI2. The phrase “geneticcomplement” is used to refer to the aggregate of nucleotide sequences,the expression of which sequences defines the phenotype of, in thepresent case, a corn plant, or a cell or tissue of that plant. An inbredgenetic complement thus represents the genetic make up of an inbredcell, tissue or plant, and a hybrid genetic complement represents thegenetic make up of a hybrid cell, tissue or plant. The invention thusprovides corn plant cells that have a genetic complement in accordancewith the inbred corn plant cells disclosed herein, and plants, seeds anddiploid plants containing such cells.

Plant genetic complements may be assessed by genetic marker profiles,and by the expression of phenotypic traits that are characteristic ofthe expression of the genetic complement, e.g., isozyme typing profiles.Thus, such corn plant cells may be defined as having an RFLP geneticmarker profile in accordance with the profile shown in Table 6, or agenetic isozyme typing profile in accordance with the profile shown inTable 7, or having both an RFLP genetic marker profile and a geneticisozyme typing profile in accordance with the profiles shown in Tables 6and 7.

In another aspect, the present invention provides hybrid geneticcomplements, as represented by corn plant cells, tissues, plants andseeds, formed by the combination of a haploid genetic complement of aninbred corn plant of the invention with a haploid genetic complement ofa second corn plant, preferably, another, distinct inbred corn plant. Inanother aspect, the present invention provides a corn plant regeneratedfrom a tissue culture that comprises a hybrid genetic complement of thisinvention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

I. Definitions

Barren Plants: Plants that are barren, i.e., lack an ear with grain, orhave an ear with only a few scattered kernels. Cg: Colletotrichumgraminicola rating. Rating times 10 is approximately equal to percenttotal plant infection. CLN: Corn Lethal Necrosis (combination of MaizeChlorotic Mottle Virus and Maize Dwarf Mosaic virus) rating: numericalratings are based on a severity scale where 1 = most resistant to 9 =susceptible. Cn: Corynebacterium nebraskense rating. Rating times 10 isapproximately equal to percent total plant infection. Cz: Cercosporazeae-maydis rating. Rating times 10 is approximately equal to percenttotal plant infection. Dgg: Diatraea grandiosella girdling rating(values are percent plants girdled and stalk lodged). Dropped Ears: Earsthat have fallen from the plant to the ground. Dsp: Diabrotica speciesroot ratings (1 = least affected to 9 = severe pruning). Ear-Attitude:The attitude or position of the ear at harvest scored as upright,horizontal, or pendant. Ear-Cob Color: The color of the cob, scored aswhite, pink, red, or brown. Ear-Cob Diameter: The average diameter ofthe cob measured at the midpoint. Ear-Cob Strength: A measure ofmechanical strength of the cobs to breakage, scored as strong or weak.Ear-Diameter: The average diameter of the ear at its midpoint. Ear-DryHusk Color: The color of the husks at harvest scored as buff, red, orpurple. Ear-Fresh Husk The color of the husks 1 to 2 weeks after Color:pollination scored as green, red, or purple. Ear-Husk Bract: The lengthof an average husk leaf scored as short, medium, or long. Ear-HuskCover: The average distance from the tip of the ear to the tip of thehusks. Minimum value no less than zero. Ear-Husk Opening: An evaluationof husk tightness at harvest scored as tight, intermediate, or open.Ear-Length: The average length of the ear. Ear-Number Per The averagenumber of ears per plant. Stalk: Ear-Shank The average number ofinternodes on the ear Internodes: shank. Ear-Shank Length: The averagelength of the ear shank. Ear-Shelling Percent: The average of theshelled grain weight divided by the sum of the shelled grain weight andcob weight for a single ear. Ear-Silk Color: The color of the silkobserved 2 to 3 days after silk emergence scored as green-yellow,yellow, pink, red, or purple. Ear-Taper (Shape): The taper or shape ofthe ear scored as conical, semi-conical, or cylindrical. Ear-Weight: Theaverage weight of an ear. Early Stand: The percent of plants that emergefrom the ground as determined in the early spring. ER: Ear rot rating(values approximate percent ear rotted). Final Stand Count: The numberof plants just prior to harvest. GDUs to Shed: The number of growingdegree units (GDUs) or heat units required for an inbred line or hybridto have approximately 50 percent of the plants shedding pollen asmeasured from time of planting. Growing degree units are calculated bythe Barger Method, where the heat units for a 24-hour period arecalculated as GDUs = [Maximum daily temperature + Minimum dailytemperature)/2] − 50. The highest maximum daily temperature used is 86degrees Fahrenheit and the lowest minimum temperature used is 50 degreesFahrenheit. GDUs to shed is then determined by summing the individualdaily values from planting date to the date of 50 percent pollen shed.GDUs to Silk: The number of growing degree units for an inbred line orhybrid to have approximately 50 percent of the plants with silkemergence as measured from time of planting. Growing degree units arecalculated by the same methodology as indicated in the GDUs to sheddefinition. Hc2: Helminthosporium carbonum race 2 rating. Rating times10 is approximately equal to percent total plant infection. Hc3:Helminthosporium carbonum race 3 rating. Rating times 10 isapproximately equal to percent total plant infection. Hm:Helminthosporium maydis race 0 rating. Rating times 10 is approximatelyequal to percent total plant infection. Ht1: Helminthosporium turcicumrace 1 rating. Rating times 10 is approximately equal to percent totalplant infection. Ht2: Helminthosporium turcicum race 2 rating. Ratingtimes 10 is approximately equal to percent total plant infection. HtG: += Presence of Ht chlorotic-lesion type resistance. Rating times 10 isapproximately equal to percent total plant infection. − = Absence of aHt chlorotic-lesion type resistance. Rating times 10 is approximatelyequal to percent total plant infection. +/− = Segregation of a Htchlorotic-lesion type resistance. Rating times 10 is approximately equalto percent total plant infection. Kernel-Aleurone The color of thealeurone scored as white, pink, Color: tan, brown, bronze, red, purple,pale purple, colorless, or variegated. Kernel-Cap Color: The color ofthe kernel cap observed at dry stage, scored as white, lemon-yellow,yellow or orange. Kernel-Endosperm The color of the endosperm scored aswhite, pale Color: yellow, or yellow. Kernel-Endosperm The type ofendosperm scored as normal, waxy, Type: or opaque. Kernel-Grade: Thepercent of kernels that are classified as rounds. Kernel-Length: Theaverage distance from the cap of the kernel to the pedicel.Kernel-Number Per The average number ofkemels in a single row. Row:Kernel-Pericarp The color of the pericarp scored as colorless, Color:red-white crown, tan, bronze, brown, light red, cherry red, orvariegated. Kernel-Row The direction of the kernel rows on the earscored Direction: as straight, slightly curved, spiral, or indistinct(scattered). Kernel-Row Number: The average number of rows of kernels ona single ear. Kernel-Side Color: The color of the kernel side observedat the dry stage, scored as white, pale yellow, yellow, orange, red, orbrown. Kernel-Thickness: The distance across the narrow side of thekernel. Kernel-Type: The type of kernel scored as dent, flint, orintermediate. Kernel-Weight: The average weight of a predeterminednumber of kernels. Kernel-Width: The distance across the flat side ofthe kernel. Kz: Kabatiella zeae rating. Rating times 10 is approx-imately equal to percent total plant infection. Leaf-Angle: Angle of theupper leaves to the stalk scored as upright (0 to 30 degrees),intermediate (30 to 60 degrees), or lax (60 to 90 degrees). Leaf-Color:The color of the leaves 1 to 2 weeks after pollin- ation scored as lightgreen, medium green, dark green, or very dark green. Leaf-Length: Theaverage length of the primary ear leaf. Leaf-Longitudinal A rating ofthe number of longitudinal creases on Creases: the leaf surface 1 to 2weeks after pollination. Creases are scored as absent, few, or many.Leaf-Marginal A rating of the waviness of the leaf margin 1 to 2 Waves:weeks after pollination. Rated as none, few, or many. Leaf-Number: Theaverage number of leaves of a mature plant. Counting begins with thecotyledonary leaf and ends with the flag leaf. Leaf-Sheath A rating ofthe level of anthocyanin in the leaf Anthocyanin: sheath 1 to 2 weeksafter pollination, scored as absent, basal-weak, basal-strong, weak orstrong. Leaf-Sheath A rating of the pubescence of the leaf sheath.Pubescence: Ratings are taken 1 to 2 weeks after pollination and scoredas light, medium, or heavy. Leaf-Width: The average width of the primaryear leaf measured at its widest point. LSS: Late season standability(values times 10 approximate percent plants lodged in disease evaluationplots). Moisture: The moisture of the grain at harvest. On1: Ostrinianubilalis 1st brood rating (1 = resistant to 9 = susceptible). On2:Ostrinia nubilalis 2nd brood rating (1 = resistant to 9 = susceptible).Relative Maturity: A maturity rating based on regression analysis. Theregression analysis is developed by utilizing check hybrids and theirpreviously established day rating versus actual harvest moistures.Harvest moisture on the hybrid in question is determined and thatmoisture value is inserted into the regression equation to yield arelative maturity. Root Lodging: Root lodging is the percentage ofplants that root lodge. A plant is counted as root lodged if a portionof the plant leans from the vertical axis by approximately 30 degrees ormore. Seedling Color: Color of leaves at the 6 to 8 leaf stage. SeedlingHeight: Plant height at the 6 to 8 leaf stage. Seedling Vigor: A visualrating of the amount of vegetative growth on a 1 to 9 scale, where 1equals best. The score is taken when the average entry in a trial is atthe fifth leaf stage. Selection Index: The selection index gives asingle measure of hy- brid's worth based on information from multipletraits. One of the traits that is almost always included is yield.Traits may be weighted accord- ing to the level of importance assignedto them. Sr: Sphacelotheca reiliana rating is actual percent infection.Stalk-Anthocyanin: A rating of the amount of anthocyanin pigmen- tationin the stalk. The stalk is rated 1 to 2 weeks after pollination asabsent, basal-weak, basal- strong, weak, or strong. Stalk-Brace Root Thecolor of the brace roots observed 1 to 2 Color: weeks after pollinationas green, red, or purple. Stalk-Diameter: The average diameter of thelowest visible internode of the stalk. Stalk-Ear Height: The averageheight of the ear measured from the ground to the point of attachment ofthe ear shank of the top developed ear to the stalk. Stalk-Internode Thedirection of the stalk internode observed after Direction: pollinationas straight or zigzag. Stalk-Internode The average length of theinternode above the Length: primary ear. Stalk Lodging: The percentageof plants that did stalk lodge. Plants are counted as stalk lodged ifthe plant is broken over or off below the ear. Stalk-Nodes With Theaverage number of nodes having brace roots Brace Roots: per plant.Stalk-Plant Height: The average height of the plant as measured from thesoil to the tip of the tassel. Stalk-Tillers: The percent ofplants thathave tillers. A tiller is defined as a secondary shoot that hasdeveloped as a tassel capable of shedding pollen. Staygreen: Staygreenis a measure of general plant health near the time of black layerformation (physio- logical maturity). It is usually recorded at the timethe ear husks of most entries within a trial have turned a mature color.Scoring is on a 1 to 9 basis where 1 equals best. STR: Stalk rot rating(values represent severity rating of 1 = 25 percent of inoculatedinternode rotted to 9 = entire stalk rotted and collapsed). SVC:Southeastern Virus Complex combination of Maize Chlorotic Dwarf Virusand Maize Dwarf Mosaic Virus) rating; numerical ratings are based on aseverity scale where 1 = most resistant to 9 = susceptible (1988reactions are largely Maize Dwarf Mosaic Virus reactions). Tassel-AntherColor: The color of the anthers at 50 percent pollen shed scored asgreen-yellow, yellow, pink, red, or purple. Tassel-Attitude: Theattitude of the tassel after pollination scored as open or compact.Tassel-Branch Angle: The angle of an average tassel branch to the mainstem of the tassel scored as upright (less than 30 degrees),intermediate (30 to 45 degrees), or lax (greater than 45 degrees).Tassel-Branch The average number of primary tassel branches. Number:Tassel-Glume Band: The closed anthocyanin band at the base of the glumescored as present or absent. Tassel-Glume Color: The color of the glumesat 50 percent shed scored as green, red, or purple. Tassel-Length: Thelength of the tassel measured from the base of the bottom tassel branchto the tassel tip. Tassel-Peduncle The average length of the tasselpeduncle, Length: measured from the base of the flag leaf to the base ofthe bottom tassel branch. Tassel-Pollen Shed: A visual rating of pollenshed determined by tapping the tassel and observing the pollen flow ofapproximately five plants per entry. Rated on a 1 to 9 scale where 9 =sterile, 1 = most pollen. Tassel-Spike Length: The length of the spikemeasured from the base of the top tassel branch to the tassel tip. TestWeight: The measure of the weight of the grain in pounds for a givenvolume (bushel) adjusted to 15.5 percent moisture. Yield: Yield of grainat harvest adjusted to 15.5 percent moisture.

II. Other Definitions

Allele is any of one or more alternative forms of a gene, all of whichalleles relate to one trait or characteristic. In a diploid cell ororganism, the two alleles of a given gene occupy corresponding loci on apair of homologous chromosomes.

Backcrossing is a process in which a breeder repeatedly crosses hybridprogeny back to one of the parents, for example, a first generationhybrid (F₁) with one of the parental genotypes of the F1 hybrid.

Chromatography is a technique wherein a mixture of dissolved substancesare bound to a solid support followed by passing a column of fluidacross the solid support and varying the composition of the fluid. Thecomponents of the mixture are separated by selective elution.

Crossing refers to the mating of two parent plants.

Cross-pollination refers to fertilization by the union of two gametesfrom different plants.

Diploid refers to a cell or organism having two sets of chromosomes.

Electrophoresis is a process by which particles suspended in a fluid aremoved under the action of an electrical field, and thereby separatedaccording to their charge and molecular weight. This method ofseparation is well known to those skilled in the art and is typicallyapplied to separating various forms of enzymes and of DNA fragmentsproduced by restriction endonucleases.

Emasculate refers to the removal of plant male sex organs.

Enzymes are organic catalysts that can exist in various forms calledisozymes.

F₁ Hybrid refers to the first generation progeny of the cross of twoplants.

Genetic Complement refers to an aggregate of nucleotide sequences, theexpression of which sequences defines the phenotype in corn plants, orcomponents of plants including cells or tissue.

Genotype refers to the genetic constitution of a cell or organism.

Haploid refers to a cell or organism having one set of the two sets ofchromosomes in a diploid.

Isozymes are one of a number of enzymes which catalyze the samereaction(s) but differ from each other, e.g., in primary structureand/or electrophoretic mobility. The differences between isozymes areunder single gene, codominant control. Consequently, electrophoreticseparation to produce band patterns can be equated to different allelesat the DNA level. Structural differences that do not alter charge cannotbe detected by this method.

Isozyme typing profile refers to a profile of band patterns of isozymesseparated by electrophoresis that can be equated to different alleles atthe DNA level.

Linkage refers to a phenomenon wherein alleles on the same chromosometend to segregate together more often than expected by chance if theirtransmission was independent.

Marker is a readily detectable phenotype, preferably inherited incodominant fashion (both alleles at a locus in a diploid heterozygoteare readily detectable), with no environmental variance component, i.e.,heritability of 1.

29MIFI2 refers to the corn plant from which seeds having ATCC AccessionNo. 209896 were obtained, as well as plants grown from those seeds.

Phenotype refers to the detectable characteristics of a cell ororganism, which characteristics are the manifestation of geneexpression.

Quantitative Trait Loci (QTL) refer to genetic loci that control to somedegree numerically representable traits that are usually continuouslydistributed.

Regeneration refers to the development of a plant from tissue culture.

RFLP genetic marker profile refers to a profile of band patterns of DNAfragment lengths typically separated by agarose gel electrophoresis,after restriction endonuclease digestion of DNA.

Self-pollination refers to the transfer of pollen from the anther to thestigma of the same plant.

Single Gene Converted (Conversion) Plant refers to plants which aredeveloped by a plant breeding technique called backcrossing whereinessentially all of the desired morphological and physiologicalcharacteristics of an inbred are recovered in addition to the singlegene transferred into the inbred via the backcrossing technique.

Tissue Culture refers to a composition comprising isolated cells of thesame or a different type or a collection of such cells organized intoparts of a plant.

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples that follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments that are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

III. Inbred Corn Plant 29MIFI2

In accordance with one aspect of the present invention, there isprovided a novel inbred corn plant, designated 29MIFI2. Inbred cornplant 29MIFI2 is a yellow, dent corn inbred that can be compared toinbred corn plant 3IIH6, a proprietary inbred of DEKALB GeneticsCorporation. 29MIFI2 differs significantly (at the 1%, 5% or 10% level)from this inbred line in several aspects (Table 1).

TABLE 1 COMPARISON OF 29MIFI2 WITH 3IIH6 BARREN DROP EHT MST PHT RTLSHED SILK STL YLD INBRED % % INCH FINAL % INCH % GDU GDU % BU/A 29MIFI20.5 0.1 24.0 49.2 16.9 59.4 0.6 1390.9 1446.2 0.8 67.6 3IIH6 0.2 0.227.2 56.9 18.8 62.3 0.8 1439.3 1476.7 1.8 80.4 DIFF 0.3 −0.1 −3.3 −7.7−2.0 −2.9 −0.2 −48.3 −30.4 −1.0 −12.8 # LOC 24 24 23 24 18 23 24 18 1824 19 P VALUE 0.32 0.85 0.00** 0.00** 0.01** 0.00** 0.86 0.00** 0.00**0.09+ 0.00** Significance Levels are indicated as: + = 10 percent, * = 5percent, ** = 1 percent. Legend Abbreviations: BARREN % = Barren Plants(percent) DROP % = Dropped Ears (percent) EHT INCH = Ear Height (inches)FINAL = Final Stand MST % = Moisture (percent) PHT INCH = Plant Height(inches) RTL % = Root Lodging (percent) SHED GDU = GDUs to Shed SILK GDU= GDUS to Silk STL % = Stalk Lodging (percent) YLD BU = Yield(bushels/acre)

A. Origin and Breeding History

Inbred plant 29MIFI2 was derived from the cross between a line derivedfrom IBC2 and 293IBZ2.

29MIFI2 shows uniformity and stability within the limits ofenvironmental influence for the traits described hereinafter in Table 2.29MIFI2 has been self-pollinated and ear-rowed a sufficient number ofgenerations with careful attention paid to uniformity of plant type toensure homozygosity and phenotypic stability. No variant traits havebeen observed or are expected in 29MIFI2.

A deposit of 2500 seeds of plant designated 29MIFI2 has been made withthe American Type Culture Collection (ATCC), 10801 University Blvd.,Manassas, Va. on May 26, 1998. Those deposited seeds have been assignedAccession No. 209896 The deposit was made in accordance with the termsand provisions of the Budapest Treaty relating to deposit ofmicroorganisms and is made for a term of at least thirty (30) years andat least five (05) years after the most recent request for thefurnishing of a sample of the deposit was received by the depository, orfor the effective term of the patent, whichever is longer, and will bereplaced if it becomes non-viable during that period.

Inbred corn plants can be reproduced by planting such inbred seeds,growing the resulting corn plants under self-pollinating orsib-pollinating conditions with adequate isolation using standardtechniques well known to an artisan skilled in the agricultural arts.Seeds can be harvested from such a plant using standard, well knownprocedures.

The origin and breeding history of inbred plant 29MIFI2 can besummarized as follows:

Summer 1986 The cross IBC2*29MIBZ2 was made. Both IBC2 and 29MIBZ2 areproprietary inbreds of DEKALB Genetics Corporation. Winter 1986-87 S0seed was grown (nursery row 29038). Summer 1987 S1 seed was grown(nursery range 205, rows 71-75). Winter 1987-88 S2 seed was grownear-to-row (nursery range 363, rows 53-80). Summer 1988 S3 seed wasgrown ear-to-row (range 217, row 87). Winter 1989 S4 ears withdesignation 1B021 & 1B022 were coded as 29MIFI2. Summer 1990 S4 seed wasgrown ear-to-row (range 729, row 46 & 47). Summer 1991 S5 seed was grownas a bulk (nursery range 724, rows 26-40 and rows 41-50).

B. Phenotypic Description

In accordance with another aspect of the present invention, there isprovided a corn plant having the physiological and morphologicalcharacteristics of corn plant 29MIFI2. A description of thephysiological and morphological characteristics of corn plant 29MIFI2 ispresented in Table 2.

TABLE 2 MORPHOLOGICAL TRAITS FOR THE 29MIFI2 PHENOTYPE VALUECHARACTERISTIC 29MIFI2 29MIBZ2 311H6 IBC2 1. STALK Diameter (width) cm.2.3 2.2 2.3 2.0 Nodes With Brace 1.2 1.3 0.7 1.5 Roots InternodeDirection Straight Straight Straight Straight Internode Length cm. 14.413.0 13.1 14.0 2. LEAF Color Med Green Med Green Med Green Med GreenLength cm. 74.7 70.9 70.5 72.3 Width cm. 8.1 8.8 8.0 8.1 Marginal WavesFew Few Few Moderate Longitudinal Creases Few Few — — 3. TASSEL Lengthcm. 37.0 32.3 35.3 36.3 Spike Length cm. 24.9 22.1 25.2 25.2 PeduncleLength cm. 8.3 4.4 7.6 12.3 Branch Number 9.1 10.0 12.9 9.0 4. EARNumber Per Stalk 1.1 1.3 1.4 1.2 Position (attitude) Pendant UprightUpright Upright Length cm. 15.0 14.7 15.6 14.4 Shape Semi-conicalSemi-conical Semi-conical Semi-conical Diameter cm. 4.0 3.8 3.9 3.7Weight gm. 105.7 101.2 107.6 98.3 Shank Length cm. 14.1 10.3 9.6 12.5Husk Bract Medium Short Short Short Husk Cover cm. 6.7 5.3 3.7 4.8 HuskColor Dry Buff Buff Buff — Cob Diameter cm. 2.3 2.2 2.1 2.2 Cob ColorRed Red Red Red Shelling Percent 83.9 79.5 83.3 81.6 5. KERNEL RowNumber Number Per Row 13.9 14.7 15.3 12.9 Row Direction 30.6 27.0 29.727.6 Type Curved Curved Curved Curved Cap Color Dent Dent — Dent SideColor Yellow Yellow Yellow — Length (depth) mm. Deep Yellow Orange — —Width mm. 9.7 10.0 10.3 9.8 Thickness 7.9 8.2 7.8 8.0 Weight of 1000 Kgm. 3.6 4.3 4.2 4.5 Endosperm Type 259.5 233.1 247.8 254.5 EndospermColor Normal Normal Normal Normal Yellow Yellow Yellow Yellow *These aretypical values. Values may vary due to environment. Other values thatare substantially equivalent are also within the scope of the invention.Substantially equivalent refers to quantitative traits that whencompared do not show statistical differences of their means.

IV. Additional Inbred Corn Plants

The inbred corn plant 3AZA1 has been employed with the corn plant of thepresent invention in order to produce an exemplary hybrids. Adescription of the physiological and morphological characteristics ofthis corn plant is presented herein at Table 3. Additional informationfor this inbred corn plant is presented in co-pending U.S. patentapplication Ser. No. 08/595,549, filed Feb. 1, 1996, the disclosure ofwhich application is specifically incorporated herein by reference.

TABLE 3 MORPHOLOGICAL TRAITS FOR THE 3AZA1 PHENOTYPE VALUECHARACTERISTIC 3AZA1 91BMA2 AQA3 FBLA 1. STALK Diameter (width) cm. 2.01.8 2.2 1.6 Anthocyanin Absent Absent Absent Absent Nodes with braceroots 2.0 1.1 1.5 0.8 Internode direction Straight Straight StraightStraight Internode length cm. 14.0 12.1 11.3 13.3 2. LEAF Angle IntermedIntermed Intermed Intermed Color Med green Med green Med green Darkgreen Number 19.0 19.0 18.5 19.7 Length cm. 66.2 65.5 67.9 68.9 Widthcm. 7.9 8.4 8.3 8.0 Sheath anthocyanin Weak — Absent Absent Sheathpubescence Medium Light Medium Light Marginal waves Absent Few Few FewLongitudinal creases Few Few Few Few 3. TASSEL Branch angle IntermedUpright Intermed Intermed Length cm. 33.0 32.0 33.0 29.2 Spike lengthcm. 24.4 23.2 23.1 23.0 Peduncle length cm. 3.6 7.7 3.6 7.1 Branchnumber 3.8 4.0 5.5 4.4 Anther color Tan — Grn-yel Tan wk anth Glumecolor Green Green Green Green Glume band Absent Absent Absent Absent 4.EAR Silk color Grn-yel Grn-yel Grn-yel Pink med anth Number per stalk1.6 1.1 1.4 1.1 Position (attitude) Pendant Upright Upright UprightLength cm. 16.4 13.5 16.3 13.2 Shape Semi-con Semi-con Semi-con Semi-conDiameter cm. 33.8 34.0 36.0 38.0 Weight gm. 89.8 92.5 93.1 99.5 Shanklength cm. 20.7 13.9 14.9 16.1 Shank internode number 6.0 6.4 7.1 6.5Husk bract Short Short Short Short Husk cover cm. 1.9 5.9 3.2 5.7 Huskcolor fresh Green Green Lt grn Green Husk color dry Buff Buff Buff BuffCob color Red Red Red Red Cob diameter cm. 17.2 2.0 2.1 2.3 Shellingpercent 85.8 82.7 85.0 80.0 5. KERNEL Row number 12.3 12.7 15.1 14.1Number per row 32.2 27.8 33.2 26.4 Row direction Curved Curved CurvedCurved Type Dent Intermed Dent Dent Cap color Yellow Yellow Lemon yelYellow Side color Orange Orange Orange Brown Length (depth) mm. 9.2 9.69.6 10.3 Width mm. 7.3 7.4 7.1 8.0 Thickness 4.2 3.5 3.8 4.4 Weight of1000 k gm. 223.8 225.3 173.7 254.0 Endosperm type Normal Normal NormalNormal Endosperm color Yellow Yellow Yellow Yellow *These are typicalvalues. Values may vary due to environment. Other values that aresubstantially equivalent are also within the scope of the invention.Substantially equivalent refers to quantitative traits that whencompared do not show statistical differences of their means.

V. Single Gene Conversions

When the term inbred corn plant is used in the context of the presentinvention, this also includes any single gene conversions of thatinbred. The term single gene converted plant as used herein refers tothose corn plants which are developed by a plant breeding techniquecalled backcrossing wherein essentially all of the desired morphologicaland physiological characteristics of an inbred are recovered in additionto the single gene transferred into the inbred via the backcrossingtechnique. Backcrossing methods can be used with the present inventionto improve or introduce a characteristic into the inbred. The termbackcrossing as used herein refers to the repeated crossing of a hybridprogeny back to one of the parental corn plants for that inbred. Theparental corn plant which contributes the gene for the desiredcharacteristic is termed the nonrecurrent or donor parent. Thisterminology refers to the fact that the nonrecurrent parent is used onetime in the backcross protocol and therefore does not recur. Theparental corn plant to which the gene or genes from the nonrecurrentparent are transferred is known as the recurrent parent as it is usedfor several rounds in the backcrossing protocol (Poehlman & Sleper,1994; Fehr, 1987). In a typical backcross protocol, the original inbredof interest (recurrent parent) is crossed to a second inbred(nonrecurrent parent) that carries the single gene of interest to betransferred. The resulting progeny from this cross are then crossedagain to the recurrent parent and the process is repeated until a cornplant is obtained wherein essentially all of the desired morphologicaland physiological characteristics of the recurrent parent are recoveredin the converted plant, in addition to the single transferred gene fromthe nonrecurrent parent.

The selection of a suitable recurrent parent is an important step for asuccessful backcrossing procedure. The goal of a backcross protocol isto alter or substitute a single trait or characteristic in the originalinbred. To accomplish this, a single gene of the recurrent inbred ismodified or substituted with the desired gene from the nonrecurrentparent, while retaining essentially all of the rest of the desiredgenetic, and therefore the desired physiological and morphological,constitution of the original inbred. The choice of the particularnonrecurrent parent will depend on the purpose of the backcross, one ofthe major purposes is to add some commercially desirable, agronomicallyimportant trait to the plant. The exact backcrossing protocol willdepend on the characteristic or trait being altered to determine anappropriate testing protocol. Although backcrossing methods aresimplified when the characteristic being transferred is a dominantallele, a recessive allele may also be transferred. In this instance itmay be necessary to introduce a test of the progeny to determine if thedesired characteristic has been successfully transferred.

Many single gene traits have been identified that are not regularlyselected for in the development of a new inbred but that can be improvedby backcrossing techniques. Single gene traits may or may not betransgenic, examples of these traits include but are not limited to,male sterility, waxy starch, herbicide resistance, resistance forbacterial, fungal, or viral disease, insect resistance, male fertility,enhanced nutritional quality, industrial usage, yield stability andyield enhancement. These genes are generally inherited through thenucleus. Some known exceptions to this are the genes for male sterility,some of which are inherited cytoplasmically, but still act as singlegene traits. Several of these single gene traits are described in U.S.Ser. No. 07/113,561. filed Aug. 25, 1993, the disclosure of which isspecifically hereby incorporated by reference.

Direct selection may be applied where the single gene acts as a dominanttrait. An example might be the herbicide resistance trait. For thisselection process, the progeny of the initial cross are sprayed with theherbicide prior to the backcrossing. The spraying eliminates any plantswhich do not have the desired herbicide resistance characteristic, andonly those plants which have the herbicide resistance gene are used inthe subsequent backcross. This process is then repeated for alladditional backcross generations.

The waxy characteristic is an example of a recessive trait. In thisexample, the progeny resulting from the first backcross generation (BC1)must be grown and selfed. A test is then run on the selfed seed from theBC1 plant to determine which BC1 plants carried the recessive gene forthe waxy trait. In other recessive traits, additional progeny testing,for example growing additional generations such as the BC1S1 may berequired to determine which plants carry the recessive gene.

VI. Origin and Breeding History of an Exemplary Single Gene ConvertedPlant

85DGD1 MLms is a single gene conversion of 85DGD1 to cytoplasmic malesterility. 85DGD1 MLms was derived using backcross methods. 85DGD1 (aproprietary inbred of DEKALB Genetics Corporation) was used as therecurrent parent and MLms, a germplasm source carrying ML cytoplasmicsterility, was used as the nonrecurrent parent. The breeding history ofthe single gene converted inbred 85DGD1 MLms can be summarized asfollows:

Hawaii Nurseries Planting Date 4-2-92 Made up S-O: Female row 585 malerow 500 Hawaii Nurseries Planting Date 7-15-92 S-O was grown and plantswere backcrossed times 85DGD1 (rows 444 ′ 443) Hawaii Nurseries PlantingDate 11-18-92 Bulked seed of the BC1 was grown and backcrossed times85DGD1 (rows V3-27 ′ V3-26) Hawaii Nurseries Planting Date 4-2-93 Bulkedseed of the BC2 was grown and backcrossed times 85DGD1 (rows 37 ′ 36)Hawaii Nurseries Planting Date 7-14-93 Bulked seed of the BC3 was grownand backcrossed times 85DGD1 (rows 99 ′ 98) Hawaii Nurseries PlantingDate 10-28-93 Bulked seed of BC4 was grown and backcrossed times 85DGD1(rows KS-63 ′ KS-62) Summer 1994 A single ear of the BC5 was grown andbackcrossed times 85DGD1 (MC94-822 ′ MC94-822-7) Winter 1994 Bulked seedof the BC6 was grown and backcrossed times 85DGD1 (3Q-1 ′ 3Q-2) Summer1995 Seed of the BC7 was bulked and named 85DGD1 MLms.

VII. Tissue Culture and in Vitro Regeneration of Corn Plants

A further aspect of the invention relates to tissue culture of cornplants designated 29MIFI2. As used herein, the term “tissue culture”indicates a composition comprising isolated cells of the same or adifferent type or a collection of such cells organized into parts of aplant. Exemplary types of tissue cultures are protoplasts, calli, plantclumps, and plant cells that are intact in plants or parts of plants,such as embryos, pollen, flowers, kernels, ears, cobs, leaves, husks,stalks, roots, root tips, anthers, silk and the like. In a preferredembodiment, tissue culture is embryos, protoplast, meristematic cells,pollen, leaves or anthers. Means for preparing and maintaining planttissue culture are well known in the art. By way of example, a tissueculture comprising organs such as tassels or anthers, has been used toproduce regenerated plants. (See, U.S. patent applications Ser. No.07/992,637, filed Dec. 18, 1992 and Ser. No. 07/995,938, filed Dec. 21,1992, now issued as U.S. Pat. No. 5,322,789, issued Jun. 21, 1994, thedisclosures of which are incorporated herein by reference).

VIII. Tassel/Anther Culture

Tassels contain anthers which in turn enclose microspores. Microsporesdevelop into pollen. For anther/microspore culture, if tassels are theplant composition, they are preferably selected at a stage when themicrospores are uninucleate, that is, include only one, rather than 2 or3 nuclei. Methods to determine the correct stage are well known to thoseskilled in the art and include mitramycin fluorescent staining (Pace etal., 1987), trypan blue (preferred) and acetocarmine squashing. Themid-uninucleate microspore stage has been found to be the developmentalstage most responsive to the subsequent methods disclosed to ultimatelyproduce plants.

Although microspore-containing plant organs such as tassels cangenerally be pretreated at any cold temperature below about 25° C., arange of 4 to 25° C. is preferred, and a range of 8 to 14° C. isparticularly preferred. Although other temperatures yield embryoids andregenerated plants, cold temperatures produce optimum response ratescompared to pretreatment at temperatures outside the preferred range.Response rate is measured as either the number of embryoids or thenumber of regenerated plants per number of microspores initiated inculture.

Although not required, when tassels are employed as the plant organ, itis generally preferred to sterilize their surface. Following surfacesterilization of the tassels, for example, with a solution of calciumhypochloride, the anthers are removed from about 70 to 150 spikelets(small portions of the tassels) and placed in a preculture orpretreatment medium. Larger or smaller amounts can be used depending onthe number of anthers.

When one elects to employ tassels directly, tassels are preferablypretreated at a cold temperature for a predefined time, preferably at10° C. for about 4 days. After pretreatment of a whole tassel at a coldtemperature, dissected anthers are further pretreated in an environmentthat diverts microspores from their developmental pathway. The functionof the preculture medium is to switch the developmental program from oneof pollen development to that of embryoid/callus development. Anembodiment of such an environment in the form of a preculture mediumincludes a sugar alcohol, for example mannitol or sorbitol, inositol orthe like. An exemplary synergistic combination is the use of mannitol ata temperature of about 1 0° C. for a period ranging from about 10 to 14days. In a preferred embodiment, 3 ml of 0.3 M mannitol combined with 50mg/l of ascorbic acid, silver nitrate and colchicine is used forincubation of anthers at 10° C. for between 10 and 14 days. Anotherembodiment is to substitute sorbitol for mannitol. The colchicineproduces chromosome doubling at this early stage. The chromosomedoubling agent is preferably only present at the preculture stage.

It is believed that the mannitol or other similar carbon structure orenvironmental stress induces starvation and functions to forcemicrospores to focus their energies on entering developmental stages.The cells are unable to use, for example, mannitol as a carbon source atthis stage. It is believed that these treatments confuse the cellscausing them to develop as embryoids and plants from microspores.Dramatic increases in development from these haploid cells, as high as25 embryoids in 10⁴ microspores, have resulted from using these methods.

In embodiments where microspores are obtained from anthers, microsporescan be released from the anthers into an isolation medium following themannitol preculture step. One method of release is by disruption of theanthers, for example, by chopping the anthers into pieces with a sharpinstrument, such as a razor blade, scalpel or Waring blender. Theresulting mixture of released microspores, anther fragments andisolation medium are then passed through a filter to separatemicrospores from anther wall fragments. An embodiment of a filter is amesh, more specifically, a nylon mesh of about 112 mm pore size. Thefiltrate which results from filtering the microspore-containing solutionis preferably relatively free of anther fragments, cell walls and otherdebris.

In a preferred embodiment, isolation of microspores is accomplished at atemperature below about 25° C. and, preferably at a temperature of lessthan about 15° C. Preferably, the isolation media, dispersing tool(e.g., razor blade) funnels, centrifuge tubes and dispersing container(e.g., petri dish) are all maintained at the reduced temperature duringisolation. The use of a precooled dispersing tool to isolate maizemicrospores has been reported (Gaillard et al., 1991).

Where appropriate and desired, the anther filtrate is then washedseveral times in isolation medium. The purpose of the washing andcentrifugation is to eliminate any toxic compounds which are containedin the non-microspore part of the filtrate and are created by thechopping process. The centrifugation is usually done at decreasing spinspeeds, for example, 1000, 750, and finally 500 rpms.

The result of the foregoing steps is the preparation of a relativelypure tissue culture suspension of microspores that are relatively freeof debris and anther remnants.

To isolate microspores, an isolation media is preferred. An isolationmedia is used to separate microspores from the anther walls whilemaintaining their viability and embryogenic potential. An illustrativeembodiment of an isolation media includes a 6 percent sucrose or maltosesolution combined with an antioxidant such as 50 mg/l of ascorbic acid,0.1 mg/l biotin and 400 mg/l of proline, combined with 10 mg/l ofnicotinic acid and 0.5 mg/l AgNO₃. In another embodiment, the biotin andproline are omitted.

An isolation media preferably has a higher antioxidant level where usedto isolate microspores from a donor plant (a plant from which a plantcomposition containing a microspore is obtained) that is field grown incontrast to greenhouse grown. A preferred level of ascorbic acid in anisolation medium is from about 50 mg/l to about 125 mg/l and, morepreferably from about 50 mg/l to about 100 mg/l.

One can find particular benefit in employing a support for themicrospores during culturing and subculturing. Any support thatmaintains the cells near the surface can be used. The microsporesuspension is layered onto a support, for example by pipetting. Thereare several types of supports which are suitable and are within thescope of the invention. An illustrative embodiment of a solid support isa TRANSWELL® culture dish. Another embodiment of a solid support fordevelopment of the microspores is a bilayer plate wherein liquid mediais on top of a solid base. Other embodiments include a mesh or amillipore filter. Preferably, a solid support is a nylon mesh in theshape of a raft. A raft is defined as an approximately circular supportmaterial which is capable of floating slightly above the bottom of atissue culture vessel, for example, a petri dish, of about a 60 or 100mm size, although any other laboratory tissue culture vessel willsuffice. In an illustrative embodiment, a raft is about 55 mm indiameter.

Culturing isolated microspores on a solid support, for example, on a 10mm pore nylon raft floating on 2.2 ml of medium in a 60 mm petri dish,prevents microspores from sinking into the liquid medium and thusavoiding low oxygen tension. These types of cell supports enable theserial transfer of the nylon raft with its associatedmicrospore/embryoids ultimately to full strength medium containingactivated charcoal and solidified with, for example, GELRITE™(solidifying agent). The charcoal is believed to absorb toxic wastes andintermediaries. The solid medium allows embryoids to mature.

The liquid medium passes through the mesh while the microspores areretained and supported at the medium-air interface. The surface tensionof the liquid medium in the petri dish causes the raft to float. Theliquid is able to pass through the mesh: consequently, the microsporesstay on top. The mesh remains on top of the total volume of liquidmedium. An advantage of the raft is to permit diffusion of nutrients tothe microspores. Use of a raft also permits transfer of the microsporesfrom dish to dish during subsequent subculture with minimal loss,disruption or disturbance of the induced embryoids that are developing.The rafts represent an advantage over the multi-welled TRANSWELL®plates, which are commercially available from COSTAR, in that thecommercial plates are expensive. Another disadvantage of these plates isthat to achieve the serial transfer of microspores to subsequent media,the membrane support with cells must be peeled off the insert in thewells. This procedure does not produce as good a yield nor as efficienttransfers, as when a mesh is used as a vehicle for cell transfer.

The culture vessels can be further defined as either (1) a bilayer 60 mmpetri plate wherein the bottom 2 ml of medium are solidified with 0.7percent agarose overlaid with 1 mm of liquid containing the microspores;(2) a nylon mesh raft wherein a wafer of nylon is floated on 1.2 ml ofmedium and 1 ml of isolated microspores is pipetted on top; or (3)TRANSWELL® plates wherein isolated microspores are pipetted ontomembrane inserts which support the microspores at the surface of 2 ml ofmedium.

After the microspores have been isolated, they are cultured in a lowstrength anther culture medium until about the 50 cell stage when theyare subcultured onto an embryoid/callus maturation medium. Medium isdefined at this stage as any combination of nutrients that permit themicrospores to develop into embryoids or callus. Many examples ofsuitable embryoid/callus promoting media are well known to those skilledin the art. These media will typically comprise mineral salts, a carbonsource, vitamins, growth regulations. A solidifying agent is optional. Apreferred embodiment of such a media is referred to by the inventor asthe “D medium” which typically includes 6N1 salts, AgNO₃ and sucrose ormaltose.

In an illustrative embodiment, 1 ml of isolated microspores are pipettedonto a 10 mm nylon raft and the raft is floated on 1.2 ml of medium “D”,containing sucrose or, preferably maltose. Both calli and embryoids candevelop. Calli are undifferentiated aggregates of cells. Type I is arelatively compact, organized and slow growing callus. Type II is asoft, friable and fast-growing one. Embryoids are aggregates exhibitingsome embryo-like structures. The embryoids are preferred for subsequentsteps to regenerating plants. Culture medium “D” is an embodiment ofmedium that follows the isolation medium and replaces it. Medium “D”promotes growth to an embryoid/callus. This medium comprises 6N1 saltsat ⅛ the strength of a basic stock solution, (major components) andminor components, plus 12 percent sucrose or, preferably 12 percentmaltose, 0.1 mg/l B1, 0.5 mg/l nicotinic acid, 400 mg/l proline and 0.5mg/l silver nitrate. Silver nitrate is believed to act as an inhibitorto the action of ethylene. Multi-cellular structures of approximately 50cells each generally arise during a period of 12 days to 3 weeks. Serialtransfer after a two week incubation period is preferred.

After the petri dish has been incubated for an appropriate period oftime, preferably two weeks, in the dark at a predefined temperature, araft bearing the dividing microspores is transferred serially to solidbased media which promotes embryo maturation. In an illustrativeembodiment, the incubation temperature is 30° C. and the mesh raftsupporting the embryoids is transferred to a 100 mm petri dishcontaining the 6N1-TGR-4P medium, an “anther culture medium.” Thismedium contains 6N1 salts, supplemented with 0.1 mg/l TIBA, 12 percentsugar (sucrose, maltose or a combination thereof), 0.5 percent activatedcharcoal, 400 mg/l proline, 0.5 mg/l B, 0.5 mg/l nicotinic acid, and 0.2percent GELRITE™ (solidifying agent) and is capable of promoting thematuration of the embryoids. Higher quality embryoids, that is,embryoids which exhibit more organized development, such as better shootmeristem formation without precocious germination were typicallyobtained with the transfer to full strength medium compared to thoseresulting from continuous culture using only, for example, the isolatedmicrospore culture (IMC) Medium “D.” The maturation process permits thepollen embryoids to develop further in route toward the eventualregeneration of plants. Serial transfer occurs to full strengthsolidified 6N1 medium using either the nylon raft, the TRANSWELL®membrane or bilayer plates, each one requiring the movement ofdeveloping embryoids to permit further development into physiologicallymore mature structures.

In an especially preferred embodiment, microspores are isolated in anisolation media comprising about 6 percent maltose, cultured for abouttwo weeks in an embryoid/calli induction medium comprising about 12percent maltose and then transferred to a solid medium comprising about12 percent sucrose.

At the point of transfer of the raft after about two weeks incubation,embryoids exist on a nylon support. The purpose of transferring the raftwith the embryoids to a solidified medium after the incubation is tofacilitate embryo maturation. Mature embryoids at this point areselected by visual inspection indicated by zygotic embryo-likedimensions and structures and are transferred to the shoot initiationmedium. It is preferred that shoots develop before roots, or that shootsand roots develop concurrently. If roots develop before shoots, plantregeneration can be impaired. To produce solidified media, the bottom ofa petri dish of approximately 100 mm is covered with about 30 ml of 0.2percent GELRITE™ (solidifying agent) solidified medium. A sequence ofregeneration media are used for whole plant formation from theembryoids.

During the regeneration process, individual embryoids are induced toform plantlets. The number of different media in the sequence can varydepending on the specific protocol used. Finally, a rooting medium isused as a prelude to transplanting to soil. When plantlets reach aheight of about 5 cm, they are then transferred to pots for furthergrowth into flowering plants in a greenhouse by methods well known tothose skilled in the art.

Plants have been produced from isolated microspore cultures by methodsdisclosed herein, including self-pollinated plants. The rate of embryoidinduction was much higher with the synergistic preculture treatmentconsisting of a combination of stress factors, including a carbon sourcewhich can be capable of inducing starvation, a cold temperature andcolchicine, than has previously been reported. An illustrativeembodiment of the synergistic combination of treatments leading to thedramatically improved response rate compared to prior methods, is atemperature of about 10° C., mannitol as a carbon source, and 0.05percent colchicine.

The inclusion of ascorbic acid, an anti-oxidant, in the isolation mediumis preferred for maintaining good microspore viability. However, thereseems to be no advantage to including mineral salts in the isolationmedium. The osmotic potential of the isolation medium was maintainedoptimally with about 6 percent sucrose, although a range of 2 percent to12 percent is within the scope of this invention.

In an embodiment of the embryoid/callus organizing media, mineral saltsconcentration in IMC Culture Media “D” is (⅛×), the concentration whichis used also in anther culture medium. The 6N1 salts major componentshave been modified to remove ammonium nitrogen. Osmotic potential in theculture medium is maintained with about 12 percent sucrose and about 400mg/l proline. Silver nitrate (0.5 mg/l) was included in the medium tomodify ethylene activity. The preculture media is further characterizedby having a pH of about 5.7 to 6.0. Silver nitrate and vitamins do notappear to be crucial to this medium but do improve the efficiency of theresponse.

Whole anther cultures can also be used in the production ofmonocotyledonous plants from a plant culture system. There are somebasic similarities of anther culture methods and microspore culturemethods with regard to the media used. A difference from isolatedmicrospore cultures is that undisrupted anthers are cultured, so that asupport, e.g., a nylon mesh support, is not needed. The first step indeveloping the anther cultures is to incubate tassels at a coldtemperature. A cold temperature is defined as less than about 25° C.More specifically, the incubation of the tassels is preferably performedat about 10° C. A range of 8 to 14° C. is also within the scope of theinvention. The anthers are then dissected from the tassels, preferablyafter surface sterilization using forceps, and placed on solidifiedmedium. An example of such a medium is designated by the inventors as6N1-TGR-P4.

The anthers are then treated with environmental conditions that arecombinations of stresses that are capable of diverting microspores fromgametogenesis to embryogenesis. It is believed that the stress effect ofsugar alcohols in the preculture medium, for example, mannitol, isproduced by inducing starvation at the predefined temperature. In oneembodiment, the incubation pretreatment is for about 14 days at 10° C.It was found that treating the anthers in addition with a carbonstructure, an illustrative embodiment being a sugar alcohol, preferably,mannitol, produces dramatically higher anther culture response rates asmeasured by the number of eventually regenerated plants, than bytreatment with either cold treatment or mannitol alone. These resultsare particularly surprising in light of teachings that cold is betterthan mannitol for these purposes, and that warmer temperatures interactwith mannitol better.

To incubate the anthers, they are floated on a preculture medium whichdiverts the microspores from gametogenesis, preferably on a mannitolcarbon structure, more specifically, 0.3 M of mannitol plus 50 mg/l ofascorbic acid. 3 ml is about the total amount in a dish, for example, atissue culture dish, more specifically, a 60 mm petri dish. Anthers areisolated from about 120 spikelets for one dish yields about 360 anthers.

Chromosome doubling agents can be used in the preculture media foranther cultures. Several techniques for doubling chromosome number(Jensen, 1974; Wan et al., 1989) have been described. Colchicine is oneof the doubling agents. However, developmental abnormalities arisingfrom in vitro cloning are further enhanced by colchicine treatments, andprevious reports indicated that colchicine is toxic to microspores. Theaddition of colchicine in increasing concentrations during mannitolpretreatment prior to anther culture and microspore culture has achievedimproved percentages.

An illustrative embodiment of the combination of a chromosome doublingagent and preculture medium is one which contains colchicine. In aspecific embodiment, the colchicine level is preferably about 0.05percent. The anthers remain in the mannitol preculture medium with theadditives for about 10 days at 10° C. Anthers are then placed onmaturation media, for example, that designated 6N1-TGR-P4, for 3 to 6weeks to induce embryoids. If the plants are to be regenerated from theembryoids, shoot regeneration medium is employed, as in the isolatedmicrospore procedure described in the previous sections. Otherregeneration media can be used sequentially to complete regeneration ofwhole plants.

The anthers are then exposed to embryoid/callus promoting medium, forexample, that designated 6N1-TGR-P4 to obtain callus or embryoids. Theembryoids are recognized by identification visually of embryonic-likestructures. At this stage, the embryoids are transferred serially to aseries of regeneration media. In an illustrative embodiment, the shootinitiation medium comprises BAP (6-benzyl-amino-purine) and NAA(naphthalene acetic acid). Regeneration protocols for isolatedmicrospore cultures and anther cultures are similar.

IX. Other Cultures and Regeneration

The present invention contemplates a corn plant regenerated from atissue culture of an inbred (e.g., 29MIFI2) or hybrid plant (e.g.,1131838) of the present invention. As is well known in the art, tissueculture of corn can be used for the in vitro regeneration of a cornplant. By way of example, a process of tissue culturing and regenerationof corn is described in European Patent Application, publication160,390, the disclosure of which is incorporated by reference. Corntissue culture procedures are also described in Green & Rhodes (1982)and Duncan et al., (1985). The study by Duncan et al. (1985) indicatesthat 97 percent of cultured plants produced calli capable ofregenerating plants. Subsequent studies have shown that both inbreds andhybrids produced 91 percent regenerable calli that produced plants.

Other studies indicate that non-traditional tissues are capable ofproducing somatic embryogenesis and plant regeneration. See, e.g.,Songstad et al. (1988); Rao et al. (1986); and Conger et al. (1987), thedisclosures of which are incorporated herein by reference. Regenerablecultures may be initiated from immature embryos as described in PCTpublication WO 95/06128, the disclosure of which is incorporated hereinby reference.

Briefly, by way of example, to regenerate a plant of this invention,cells are selected following growth in culture. Where employed, culturedcells are preferably grown either on solid supports or in the form ofliquid suspensions as set forth above. In either instance, nutrients areprovided to the cells in the form of media, and environmental conditionsare controlled. There are many types of tissue culture media comprisingamino acids, salts, sugars, hormones and vitamins. Most of the mediaemployed to regenerate inbred and hybrid plants have some similarcomponents, the media differ in the composition and proportions of theiringredients depending on the particular application envisioned. Forexample, various cell types usually grow in more than one type of media,but exhibit different growth rates and different morphologies, dependingon the growth media. In some media, cells survive but do not divide.Various types of media suitable for culture of plant cells have beenpreviously described and discussed above.

An exemplary embodiment for culturing recipient corn cells in suspensioncultures includes using embryogenic cells in Type II (Armstrong & Green,1985; Gordon-Kamm et al., 1990) callus, selecting for small (10 to 30 m)isodiametric, cytoplasmically dense cells, growing the cells insuspension cultures with hormone containing media, subculturing into aprogression of media to facilitate development of shoots and roots, andfinally, hardening the plant and readying it metabolically for growth insoil.

Meristematic cells (i.e., plant cells capable of continual cell divisionand characterized by an undifferentiated cytological appearance,normally found at growing points or tissues in plants such as root tips,stem apices, lateral buds, etc.) can be cultured.

Embryogenic calli are produced essentially as described in PCTPublication WO 95/06128. Specifically, inbred plants or plants fromhybrids produced from crossing an inbred of the present invention withanother inbred are grown to flowering in a greenhouse. Explants from atleast one of the following F₁ tissues: the immature tassel tissue,intercalary meristems and leaf bases, apical meristems, immature earsand immature embryos are placed in an initiation medium which contain MSsalts, supplemented with thiamine, agar, and sucrose. Cultures areincubated in the dark at about 23° C. All culture manipulations andselections are performed with the aid of a dissecting microscope.

After about 5 to 7 days, cellular outgrowths are observed from thesurface of the explants. After about 7 to 21 days, the outgrowths aresubcultured by placing them into fresh medium of the same composition.Some of the intact immature embryo explants are placed on fresh medium.Several subcultures later (after about 2 to 3 months) enough material ispresent from explants for subdivision of these embryogenic calli intotwo or more pieces.

Callus pieces from different explants are not mixed. After furthergrowth and subculture (about 6 months after embryogenic callusinitiation), there are usually between 1 and 100 pieces derivedultimately from each selected explant. During this time of cultureexpansion, a characteristic embryogenic culture morphology develops as aresult of careful selection at each subculture. Any organized structuresresembling roots or root primordia are discarded. Material known fromexperience to lack the capacity for sustained growth is also discarded(translucent, watery, embryogenic structures). Structures with a firmconsistency resembling at least in part the scutelum of the in vivoembryo are selected.

The callus is maintained on agar-solidified MS or N6-type media. Apreferred hormone is 2,4-D. A second preferred hormone is dicamba.Visual selection of embryo-like structures is done to obtainsubcultures. Transfer of material other than that displaying embryogenicmorphology results in loss of the ability to recover whole plants fromthe callus.

Cell suspensions are prepared from the calli by selecting cellpopulations that appear homogeneous macroscopically. A portion of thefriable, rapidly growing embryogenic calli is inoculated into MS or N6Medium containing 2,4-D or dicamba. The calli in medium are incubated atabout 27° C. on a gyrotary shaker in the dark or in the presence of lowlight. The resultant suspension culture is transferred about once everythree to seven days, preferably every three to four days, by takingabout 5 to 10 ml of the culture and introducing this inoculum into freshmedium of the composition listed above.

For regeneration, embryos which appear on the callus surface areselected and regenerated into whole plants by transferring theembryogenic structures into a sequence of solidified media which includedecreasing concentrations of 2,4-D or other auxins. Other hormones whichcan be used in culture media include dicamba, NAA, ABA, BAP, and 2-NCA.The reduction is relative to the concentration used in culturemaintenance media. Plantlets are regenerated from these embryos bytransfer to a hormone-free medium, subsequently transferred to soil, andgrown to maturity.

Progeny are produced by taking pollen and selfing, backcrossing orsibling regenerated plants by methods well known to those skilled in thearts. Seeds are collected from the regenerated plants.

X. Processes of Preparing Corn Plants and the Corn Plants Produced bySuch Crosses

The present invention also provides a process of preparing a novel cornplant and a corn plant produced by such a process. In accordance withsuch a process, a first parent corn plant is crossed with a secondparent corn plant wherein at least one of the first and second cornplants is the inbred corn plant 29MIFI2. In one embodiment, a corn plantprepared by such a process is a first generation F, hybrid corn plantprepared by a process wherein both the first and second parent cornplants are inbred corn plants.

Corn plants (Zea mays L.) can be crossed by either natural or mechanicaltechniques. Natural pollination occurs in corn when wind blows pollenfrom the tassels to the silks that protrude from the tops of theincipient ears. Mechanical pollination can be effected either bycontrolling the types of pollen that can blow onto the silks or bypollinating by hand.

In a preferred embodiment, crossing comprises the steps of:

(a) planting in pollinating proximity seeds of a first and a secondparent corn plant, and preferably, seeds of a first inbred corn plantand a second, distinct inbred corn plant;

(b) cultivating or growing the seeds of the first and second parent cornplants into plants that bear flowers;

(c) emasculating flowers of either the first or second parent cornplant, i.e., treating the flowers so as to prevent pollen production, inorder to produce an emasculated parent corn plant;

(d) allowing natural cross-pollination to occur between the first andsecond parent corn plant;

(e) harvesting seeds produced on the emasculated parent corn plant; and,where desired,

(f) growing the harvested seed into a corn plant, or preferably, ahybrid corn plant.

Parental plants are planted in pollinating proximity to each other byplanting the parental plants in alternating rows, in blocks or in anyother convenient planting pattern. Plants of both parental parents arecultivated and allowed to grow until the time of flowering.Advantageously, during this growth stage, plants are in general treatedwith fertilizer and/or other agricultural chemicals as consideredappropriate by the grower.

At the time of flowering, in the event that plant 29MIFI2, is employedas the male parent, the tassels of the other parental plant are removedfrom all plants employed as the female parental plant. The detasselingcan be achieved manually but also can be done by machine, if desired.

The plants are then allowed to continue to grow and naturalcross-pollination occurs as a result of the action of wind, which isnormal in the pollination of grasses, including corn. As a result of theemasculation of the female parent plant, all the pollen from the maleparent plant 29MIFI2 is available for pollination because tassels, andthereby pollen bearing flowering parts, have been previously removedfrom all plants of the inbred plant being used as the female in thehybridization. Of course, during this hybridization procedure, theparental varieties are grown such that they are isolated from other cornfields to minimize or prevent any accidental contamination of pollenfrom foreign sources. These isolation techniques are well within theskill of those skilled in this art.

Both parental inbred plants of corn may be allowed to continue to growuntil maturity or the male rows may be destroyed after flowering iscomplete. Only the ears from the female inbred parental plants areharvested to obtain seeds of a novel F₁ hybrid. The novel F₁ hybrid seedproduced can then be planted in a subsequent growing season with thedesirable characteristics in terms of F₁ hybrid corn plants providingimproved grain yields and the other desirable characteristics disclosedherein, being achieved.

Alternatively, in another embodiment, both first and second parent cornplants can come from the same inbred corn plant, i.e., from the inbreddesignated 29MIFI2. Thus, any corn plant produced using a process of thepresent invention and inbred corn plant 29MIFI2, is contemplated by thisinvention. As used herein, crossing can mean selfing, backcrossing,crossing to another or the same inbred, crossing to populations, and thelike. All corn plants produced using the present inbred corn plant29MIFI2 as a parent are within the scope of this invention.

The utility of the inbred plant 29MIFI2 also extends to crosses withother species. Commonly, suitable species will be of the familyGraminaceae, and especially of the genera Zea, Tripsacum, Coix,Schlerachne, Polytoca, Chionachne, and Trilobachne, of the tribeMaydeae. Of these, Zea and Tripsacum, are most preferred. Potentiallysuitable for crosses with 29MIFI2 can be the various varieties of grainsorghum, Sorghum bicolor (L.) Moench.

A. F₁ Hybrid Corn Plant and Seed Production

Where the inbred corn plant 29MIFI2 is crossed with another, different,corn inbred, a first generation (F₁) corn hybrid plant is produced. Botha F₁ hybrid corn plant and a seed of that F₁ hybrid corn plant arecontemplated as aspects of the present invention.

Inbred 29MIFI2 has been used to prepare an F₁ hybrid corn plant,designated 1131838.

The goal of a process of producing an F₁ hybrid is to manipulate thegenetic complement of corn to generate new combinations of genes whichinteract to yield new or improved traits (phenotypic characteristics). Aprocess of producing an F₁ hybrid typically begins with the productionof one or more inbred plants. Those plants are produced by repeatedcrossing of ancestrally related corn plants to try and concentratecertain genes within the inbred plants. The production of inbred 29MIFI2has been set forth hereinbefore.

Corn has a diploid phase which means two conditions of a gene (twoalleles) occupy each locus (position on a chromosome). If the allelesare the same at a locus, there is said to be homozygosity. If they aredifferent, there is said to be heterozygosity. In a completely inbredplant, all loci are homozygous. Because many loci when homozygous aredeleterious to the plant, in particular leading to reduced vigor, lesskernels, weak and/or poor growth, production of inbred plants is anunpredictable and arduous process. Under some conditions, heterozygousadvantage at some loci effectively bars perpetuation of homozygosity.

Inbreeding requires coddling and sophisticated manipulation by humanbreeders. Even in the extremely unlikely event inbreeding rather thancrossbreeding occurred in natural corn, achievement of completeinbreeding cannot be expected in nature due to well known deleteriouseffects of homozygosity and the large number of generations the plantwould have to breed in isolation. The reason for the breeder to createinbred plants is to have a known reservoir of genes whose gametictransmission is at least somewhat predictable.

The development of inbred plants generally requires at least about 5 to7 generations of selfing. Inbred plants are then cross-bred in anattempt to develop improved F₁ hybrids. Hybrids are then screened andevaluated in small scale field trials. Typically, about 10 to 15phenotypic traits, selected for their potential commercial value, aremeasured. A selection index of the most commercially important traits isused to help evaluate hybrids. FACT, an acronym for Field AnalysisComparison Trial (strip trials), is an on-farm testing program employedby DEKALB Plant Genetics to perform the final evaluation of thecommercial potential of a product.

During the next several years, a progressive elimination of hybridsoccurs based on more detailed evaluation of their phenotype. Eventually,strip trials (FACT) are conducted to formally compare the experimentalhybrids being developed with other hybrids, some of which werepreviously developed and generally are commercially successful. That is,comparisons of experimental hybrids are made to competitive hybrids todetermine if there was any advantage to further commercial developmentof the experimental hybrids. Examples of such comparisons are presentedin Section B, hereinbelow.

When the inbred parental plant 29MIFI2 is crossed with another inbredplant to yield a hybrid (such as the hybrid 1131838), the originalinbred can serve as either the maternal or paternal plant. For manycrosses, the outcome is the same regardless of the assigned sex of theparental plants.

However, there is often one of the parental plants that is preferred asthe maternal plant because of increased seed yield and productioncharacteristics. Some plants produce tighter ear husks leading to moreloss, for example due to rot. There can be delays in silk formationwhich deleteriously affect timing of the reproductive cycle for a pairof parental inbreds. Seed coat characteristics can be preferable in oneplant. Pollen can be shed better by one plant. Other variables can alsoaffect preferred sexual assignment of a particular cross.

B. F₁ Hybrid Comparisons

As mentioned in Section A, hybrids are progressively eliminatedfollowing detailed evaluations of their phenotype, including formalcomparisons with other commercially successful hybrids. Strip trials areused to compare the phenotypes of hybrids grown in as many environmentsas possible. They are performed in many environments to assess overallperformance of the new hybrids and to select optimum growing conditions.Because the corn is grown in close proximity, environmental factors thataffect gene expression, such as moisture, temperature, sunlight andpests, are minimized. For a decision to be made that a hybrid is worthmaking commercially available, it is not necessary that the hybrid bebetter than all other hybrids. Rather, significant improvements must beshown in at least some traits that would create improvements in someniches.

Examples of such comparative data are set forth hereinbelow in Table 4,which presents a comparison of performance data for the hybrid 1131838versus selected hybrids of commercial value (DK343 and DK363). 1131838has 29MIFI2 as one inbred parent.

All the data in Table 4 represents results across years and locationsfor research and/or strip trials. The “NTEST” represents the number ofpaired observations in designated tests at locations around the UnitedStates.

TABLE 4 COMPARATIVE DATA FOR 1131838 N SI YLD MST STL RTL DRP FLSTD SVELSTD PHT EHT BAR SG TST ESTR HYBD TEST % C BU PTS % % % % M RAT % MINCH INCH % RAT LBS FGDU DAYS 1131838 R 109.7  138.9 21.5 5.2 1.1 0.1 99.7 3.5  99.2 90.0 38.7 3.6 53.8 1175.0 85.8 134 DK343 97.5 124.5 21.13.9 1.9 0.1 100.6 3.7 102.1 84.5 39.1 5.7 54.3 1444.0 84.8 DEV 12.2 14.4  0.4 1.3 −0.8  0.0  −0.9 −0.2   −2.9  5.5 −0.3 −2.1  −0.5  31.2 0.9 ** ** ** ** * + ** ** ** ** ** ** ** F 36 101.6  121.7 22.7 5.7 0.30.4 101.8 53.5 86.9 99.3 116.8 22.4 2.7 0.3 0.3 101.5 54.6 86.0  2.4 4.9  0.3 3.1 0.0 0.2  0.2 −1.1  0.9 * * ** 1131838 R 75 115.2  140.421.0 5.8 0.9 0.1 100.1 3.1 100.9 89.9 38.9 3.1 54.0 1201.0 86.0 DK36389.0 116.2 20.1 9.2 2.5 0.1 100.5 3.5 101.1 85.2 39.0 5.1 53.5 1203.085.2 DEV 26.2  24.2  0.9 −3.4  −1.7  0.0  −0.3 −0.4   −0.2  4.6 −0.1−1.9   0.6  −2.0  0.8 ** ** ** ** * ** ** ** ** ** Significance levelsare indicated as: + = 10 percent, * = 5 percent, ** = 1 percent. LEGENDABBREVIATIONS: HYBD = Hybrid TEST = Research/FACT SI % C = SelectionIndex (percent of check) YLD BU = Yield (bushels/acre) MST PTS =Moisture STL % = Stalk Lodging (percent) RTL % = Root Lodging (percent)DRP % = Dropped Ears (percent) FLSTD % M = Final Stand (percent of testmean) SV RAT = Seedling Vigor Rating ELSTD % M = Early Stand (percent oftest mean) PHT INCH = Plant Height (inches) EHT INCH = Ear Height(inches) BAR % = Barrren Plants (percent) SG RAT = Staygreen Rating TSTLBS = Test Weight (pounds) FGDU = GDUs to Shed ESTR DAYS = EstimatedRelative Maturity (days)

As can be seen in Table 4, the hybrid 1131838 has significantly higheryield when compared to successful commercial hybrids. Significantdifferences are also shown in Table 4 for many other traits.

C. Physical Description of F₁ Hybrids

The present invention also provides F₁ hybrid corn plants derived fromthe corn plant 29MIFI2. Physical characteristics of exemplary hybridsare set forth in Table 5, which concerns 1131838, which has 29MIFI2 asone inbred parent. An explanation of terms used in Table 5 can be foundin the Definitions, set forth hereinabove.

TABLE 5 MORPHOLOGICAL TRAITS FOR THE 1131838 PHENOTYPE YEAR OF DATA:1996 CHARACTERISTIC VALUE 1. STALK Diameter (width) cm. 2.3 AnthocyaninWeak Nodes With Brace Roots 1.6 Brace Root Color Faint InternodeDirection Straight Internode Length cm. 18.3  2. LEAF Color Med GreenLength cm. 83.2  Width cm. 9.3 Sheath Anthocyanin Weak Sheath PubescenceHeavy Marginal Waves Medium Longitudinal Creases Many 3. TASSEL AttitudeCompact Length cm. 46.9  Spike Length cm. 31.8  Peduncle Length cm. 6.4Branch Number 5.9 Anther Color Tan Glume Color Green Glume Band Absent4. EAR Silk Color Tan Number Per Stalk 1.0 Position (attitude) PendantLength cm. 18.3  Shape Semi-conical Diameter cm. 4.1 Weight gm. 162.7 Shank Length cm. 20.8  Husk Bract Medium Husk Cover cm. 1.2 Husk OpeningOpen Husk Color Fresh Green Husk Color Dry Buff Cob Diameter cm. 2.2 CobColor Red Shelling Percent 87.8  5. KERNEL Row Number 14.2  Number PerRow 39.7  Row Direction Curved Type Dent Cap Color Yellow Side ColorOrange Length (depth) mm. 10.8  Width mm. 7.6 Thickness 4.2 Weight of1000K gm. 276.0  Endosperm Type Normal Endosperm Color Yellow * Theseare typical values. Values may vary due to environment. Other valuesthat are substantially equivalent are also within the scope of theinvention. Substantialy equivalent refers to quantitative traits thatwhen compared do not show statistical differences of their means.

XI. Genetic Complements

In another aspect, the present invention provides a genetic complementof a plant of this invention. In one embodiment, therefore, the presentinvention contemplates an inbred genetic complement of the inbred cornplant designated 29MIFI2. In another embodiment, the present inventioncontemplates a hybrid genetic complement formed by the combination of ahaploid genetic complement from 29MIFI2 and another haploid geneticcomplement. Means for determining a genetic complement are well-known inthe art.

As used herein, the phrase “genetic complement” means an aggregate ofnucleotide sequences, the expression of which sequences defines thephenotype of a corn plant or a cell or tissue of that plant. By way ofexample, a corn plant is genotyped to determine the array of theinherited markers it possesses. Markers are alleles at a single locus.They are preferably inherited in codominant fashion so that the presenceof both alleles at a diploid locus is readily detectable, and they arefree of environmental variation, i.e., their heritability is 1. Thisgenotyping is preferably performed on at least one generation of thedescendant plant for which the numerical value of the quantitative traitor traits of interest are also determined. The array of single locusgenotypes is expressed as a profile of marker alleles, two at eachlocus. The marker allelic composition of each locus can be eitherhomozygous or heterozygous. Homozygosity is a condition where bothalleles at a locus are characterized by the same nucleotide sequence.Heterozygosity refers to different conditions of the gene at a locus.Markers that are used for purposes of this invention include restrictionfragment length polymorphisms (RFLPs) and isozymes.

A plant genetic complement can be defined by genetic marker profilesthat can be considered “fingerprints” of a genetic complement. Forpurposes of this invention, markers are preferably distributed evenlythroughout thc genome to increase the likelihood they will be near aquantitative trait loci (QTL) of interest (e.g., in tomatoes,Helentjaris et al., U.S. Pat. No. 5,385,835, Nienhuis et al., 1987).These profiles are partial projections of a sample of genes. One of theuses of markers in general is to exclude, or alternatively include,potential parents as contributing to offspring.

Phenotypic traits characteristic of the expression of a geneticcomplement of this invention are distinguishable by electrophoreticseparation of DNA sequences cleaved by various restrictionendonucleases. Those traits (genetic markers) are termed RFLP's(restriction fragment length polymorphisms).

Restriction fragment length polymorphisms (RFLPs) are geneticdifferences detectable by DNA fragment lengths, typically revealed byagarose gel electrophoresis, after restriction endonuclease digestion ofDNA. There are large numbers of restriction endonucleases available,characterized by their nucleotide cleavage sites and their source, e.g.,Eco RI. Variations in RFLPs result from nucleotide base pair differenceswhich alter the cleavage sites of the restriction endonucleases,yielding different sized fragments.

Means for performing RFLP analyses are well known in the art.Restriction fragment length polymorphism analyses reported herein wereconducted by Linkage Genetics. This service is available to the publicon a contractual basis. Probes were prepared to the fragment sequences,these probes being complementary to the sequences thereby being capableof hybridizing to them under appropriate conditions well known to thoseskilled in the art. These probes were labeled with radioactive isotopesor fluorescent dyes for ease of detection. After the fragments wereseparated by size, they were identified by the probes. Hybridizationwith a unique cloned sequence permits the identification of a specificchromosomal region (locus). Because all alleles at a locus aredetectable, RFLPs are codominant alleles, thereby satisfying a criteriafor a genetic marker. They differ from some other types of markers,e.g., from isozymes, in that they reflect the primary DNA sequence, theyare not products of transcription or translation. Furthermore, differentRFLP genetic marker profiles result from different arrays of restrictionendonucleases.

The RFLP genetic marker profile of each of the parental inbreds andexemplary resultant hybrids were determined. Because an inbred isessentially homozygous at all relevant loci, an inbred should, in almostall cases, have only one allele at each locus. In contrast, a diploidgenetic marker profile of a hybrid should be the sum of those parents,e.g., if one inbred parent had the allele A at a particular locus, andthe other inbred parent had B, the hybrid is AB by inference. Subsequentgenerations of progeny produced by selection and breeding areanticipated to be of genotype A, B, or AB for that locus position. Whenthe F1 plant is used to produce an inbred, the locus should be either Aor B for that position. Surprisingly, it has been observed that incertain instances, novel RFLP genotypes arise during the breedingprocess. For example, a genotype of C is observed at a particular locusposition from the cross of parental inbreds with A and B at that locus.Such a novel RFLP genotype is observed for the 29MIFI2, at least, forthe RFLP markers M1447H, M3457E and M6280H, as shown in Table 6. Thesenovel RFLP markers further define the 29MIFI2 inbred from the parentalinbreds from which it was derived.

An RFLP genetic marker profile of 29MIFI2 is presented in Table 6.

TABLE 6 RFLP PROFILE OF 29MIFI2 CHARACTERISTIC 29MIFI2 29MIBZ2 3IIH6IBC2 M0306H A A A A M0445E A A A A M1120S E E E E M1234H I I I I M1401EA A C A M1406H B B B B M1447H B D B A M1B725E B H B B M2239H A — A —M2297H A A B C M2298E C C C C M2402H E E C E M3212S A — C — M3257S A — A— M3296H C C — C M3432H D D A D M3446S C — F — M3457E A C C C M4386H D DD D M4396H H H H H M4444H D A A D MM4451H B B B B M4UMC19H A A A AM4UMC31E B B B — M4UMC31S D — D — M5213S B B A B M5288S A — A — M5295E CC — C M5409H C C C C M5UMC95H D D B B M6223E B C C B M6252H A A A AM6280H G C C C M6373E E E E E M7263E B B A B M7392S C — A — M7455H B A BB M8107S E B A E M8110S A A A A M8114E B B B B M8268H B A L B M8UMC48E CC A C M9209E A A A A M9211E C C C C M9266S A — B — M9B713S A A A AM2UMC34H E — D — M9UMC94H E — B — M3UM121X C — A — M0UMC13O C — C —*Probes used to detect RFLPs are from Linkage Genetics, 1515 West 2200South, Suite C, Salt Lake City, Utah 84119.

Another aspect of this invention is a plant genetic complementcharacterized by a genetic isozyme typing profile. Isozymes are forms ofproteins that are distinguishable, for example, on starch gelelectrophoresis, usually by charge and/or molecular weight. T hetechniques and nomenclature for isozyme analysis are described in,Stuber et al. (1988), which is incorporated by reference.

A standard set of loci can be used as a reference set. Comparativeanalysis of these loci is used to compare the purity of hybrid seeds, toassess the increased variability in hybrids compared to inbreds, and todetermine the identity of seeds, plants, and plant parts. In thisrespect, an isozyme reference set can be used to develop genotypic“fingerprints.”

Table 7 lists the identifying numbers of the alleles at isozyme locitypes, and represents the exemplary genetic isozyme typing profile for29MIFI2.

TABLE 7 ISOZYME PROFILE OF 29MIFI2 ISOZYME ALLELE LOCUS 29MIFI2 29MIBZ23IIH6 IBC2 Acph1 2 2 2 2 Adh1 4 4 — 4 Cat3 9 9 9 9 Got1 4 4 4 4 Got2 2 24 2 Got3 4 4 4 4 Idh1 4 4 4 4 Idh2 4 6 6 4 Mdh1 6 6 6 6 Mdh2  6* 3.5  3.5     6* (5K)    3.5 (1K) Mdh3 16  16 16  16  Mdh4 12  12 12  12 Mdh5 12  12 12  12  Pgm1 9 9 9 9 Pgm2 4 4 4 4 6Pgd1   3.8 3.8   3.8  3.8 6Pgd2 5 5 5 5 Phi1 4 4 4 4 *Allele is probably a 6, but nullcannot be ruled out.

The present invention also contemplates a hybrid genetic complementformed by the combination of a haploid genetic complement of the cornplant 29MIFI2 with a haploid genetic complement of a second corn plant.Means for combining a haploid genetic complement from the foregoinginbred with another haploid genetic complement can be any methodhereinbefore for producing a hybrid plant from 29MIFI2. It is alsocontemplated that a hybrid genetic complement can be prepared using invitro regeneration of a tissue culture of a hybrid plant of thisinvention.

A hybrid genetic complement contained in the seed of a hybrid derivedfrom 29MIFI2 is a further aspect of this invention. Exemplary hybridgenetic complements are the genetic complements of the hybrid 1131838.

Table 8 shows the identifying numbers of the alleles for the hybrid1131838, which are exemplary RFLP genetic marker profiles for hybridsderived from the inbred of the present invention. Table 8 concerns1131838, which has 29MIFI2 as one inbred parent.

TABLE 8 RFLP PROFILE FOR 1131838 Probe/Enzyme Combination Allelic PairM0445E AB M1120S DE M1234H EI M1401E AC M1406H BB M1447H BE M1B725E BCM2239H AC M2297H AE M2298E CC M2402H EE M3257S AB M3296H CD M3432H ADM3446S CC M3457E AE M4386H DA M4396E FH M4444H AD M4451H BB M4UMC19H AAM4UMC31S BD M5213S BB M5295E CC M5409H AC M5UMC95H BD M6223E BC M6252HAD M6280H AG M6373E AE M7263E AB M7392S BC M7455H BC M8110S AC M8114E BEM8268H BB M8585H AA M8B2369S BD M8UMC48E CC M9209E AA M9211E CF M9266SAC M9B713S AB M2UMC34H DE M9UMC94H BE M3UM121X CC *Probes used to detectRFLPs are from Linkage Genetics, 1515 West 2200 South, Suite C, SaltLake City, Utah 84119.

The exemplary hybrid genetic complements of hybrid 1131838 may also beassessed by genetic isozyme typing profiles using a standard set of locias a reference set, using, e.g., the same, or a different, set of locito those described above. Table 9 lists the identifying numbers of thealleles at isozyme loci types and presents the exemplary genetic isozymetyping profile for the hybrid 1131838, which is an exemplary hybridderived from the inbred of the present invention. Table 9 concerns1131838, which has 29MIFI2 as one inbred parent.

TABLE 9 ISOZYME GENOTYPE FOR HYBRID 1131838 Locus Isozyme Alleles Acph12/4 Adh1 4 Cat3 9 Got3 4 Got2 2/4 Got1 4 Idh1 4 Idh2 4/6 Mdh1 6 Mdh2  6*Mdh3 16  Mdh4 12  Mdh5 12  Pgm1 9 Pgm2 4 6-Pgd1   3.8 6-Pgd2 5 Phi1 4*Allele is probably a 6, but a null cannot be ruled out.

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of the foregoing illustrative embodiments, itwill be apparent to those of skill in the art that variations, changes,modifications and alterations may be applied to the composition,methods, and in the steps or in the sequence of steps of the methodsdescribed herein, without departing from the true concept, spirit andscope of the invention. More specifically, it will be apparent thatcertain agents that are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

Armstrong & Green, “Establishment and Maintenance of Friable EmbryogenicMaize Callus and the Involvement of L-Proline,” Planta, 164:207-214,1985.

Conger et al., “Somatic Embryogenesis from Cultured Leaf Segments of ZeaMays,” Plant Cell Reports, 6:345-347, 1987.

Duncan et al., “The Production of Callus Capable of Plant Regenerationfrom Immature Embryos of Numerous Zea Mays Genotypes,” Planta,165:322-332, 1985.

Fehr (ed.), Principles of Cultivar Development, Vol. 1: Theory andTechnique, pp. 360-376, 1987.

Gaillard et al., “Optimization of Maize Microspore Isolation and CultureCondition for Reliable Plant Regeneration,” Plant Cell Reports,10(2):55, 1991.

Gordon-Kamm et al., “Transformation of Maize Cells and Regeneration ofFertile Transgenic Plants,” The Plant Cell, 6:603-618, 1990.

Green & Rhodes, “Plant Regeneration in Tissue Cultures of Maize,” Maizefor Biological Research, Plant Molecular Biology Association, pp.367-372, 1982.

Jensen, “Chromosome Doubling Techniques in Haploids,” Haploids andHigher Plants—Advances and Potentials, Proceedings of the FirstInternational Symposium, University of Guelph, Jun. 10-14, 1974.

Nienhuis et al., “Restriction Fragment length Polymorphism Analysis ofLoci Associated with Insect Resistance in Tomato,” Crop Science,27:797-803, 1987.

Pace et al., “Anther Culture of Maize and the Visualization ofEmbryogenic Microspores by Fluorescent Microscopy,” Theoretical andApplied Genetics, 73:863-869, 1987.

Poehlman & Sleper (eds), Breeding Field Crops, 4th Ed., pp. 172-175,1995.

Rao et al., “Somatic Embryogenesis in Glume Callus Cultures,” MaizeGenetics Cooperation Newsletter, Vol. 60, 1986.

Songstad et al. “Effect of 1-Aminocyclopropate-1-Carboxylic Acid, SilverNitrate, and Norbornadiene on Plant Regeneration from Maize CallusCultures,” Plant Cell Reports, 7:262-265, 1988.

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What is claimed is:
 1. The inbred corn seed designated 29MIFI2, a sampleof said seed having been deposited under ATCC Accession No.
 209896. 2.The inbred corn seed of claim 1, further defined as an essentiallyhomogeneous population of inbred corn plant designated 29MIFI2, a sampleof said seed having been deposited under ATCC Accession No.
 209896. 3.The inbred corn seed of claim 1, further defined as essentially freefrom hybrid seed.
 4. An inbred corn plant produced by growing the seedof an inbred corn plant designated 29MIFI2, a sample of said seed havingbeen deposited under ATCC Accession No.
 209896. 5. Pollen of the plantof claim
 4. 6. An ovule of the plant of claim
 4. 7. An essentiallyhomogeneous population of corn plants produced by growing the seed of aninbred corn plant designated 29MIFI2, a sample of said seed having beendeposited under ATCC Accession No.
 209896. 8. A corn plant having allthe physiological and morphological characteristics of the inbred cornplant 29MIFI2, a sample of the seed of said inbred corn plant 29MIFI2having been deposited under ATCC Accession No.
 209896. 9. The corn plantof claim 8, further comprising a cytoplasmic factor conferring malesterility.
 10. A tissue culture of regenerable cells of inbred cornplant 29MIFI2, wherein the tissue regenerates plants having all thephysiological and morphological characteristics of corn plant 29MIFI2 asample of the seed of said corn plant 29MIFI2 having been depositedunder ATCC Accession No.
 209896. 11. The tissue culture of claim 10,wherein the regenerable cells are embryos, meristematic cells, pollen,leaves, anthers, roots, root tips, silk, flowers, kernels, ears, cobs,husks, stalks, or protoplasts or callus derived therefrom.
 12. A cornplant regenerated from the tissue culture of claim 10, having all thephysiological and morphological characteristics of corn plant 29MIFI2, asample of the seed of said corn plant 29MIFI2 having been depositedunder ATCC Accession No.
 209896. 13. An inbred corn plant cell of thecorn plant of claim 8 having: (a) an RFLP genetic marker profile inaccordance with the profile shown in Table 6; or (b) a genetic isozymetyping profile in accordance with the profile shown in Table
 7. 14. Theinbred corn plant cell of claim 13, having an RFLP genetic markerprofile in accordance with the profile shown in Table
 6. 15. The inbredcorn plant cell of claim 13, having a genetic isozyme typing profile inaccordance with the profile shown in Table
 7. 16. The inbred corn plantcell of claim 13, having an RFLP genetic marker profile and a geneticisozyme typing profile in accordance with the profiles shown in Tables 6and
 7. 17. The inbred corn plant cell of claim 13, located within a cornplant or seed.
 18. The corn plant of claim 8, further defined as having:(c ) an RFLP genetic marker profile in accordance with the profile shownin Table 6; or (d) a genetic isozyme typing profile in accordance withthe profile shown in Table
 7. 19. The inbred corn plant of claim 18,having an RFLP genetic marker profile in accordance with the profileshown in Table
 6. 20. The inbred corn plant of claim 18, having agenetic isozyme typing profile in accordance with the profile shown inTable
 7. 21. The inbred corn plant of claim 18, having an RFLP geneticmarker profile and a genetic isozyme typing profile in accordance withthe profiles shown in Tables 6 and
 7. 22. A process of preparing cornseed, comprising crossing a first parent corn plant with a second parentcorn plant, wherein said first or second corn plant is the inbred cornplant 29MMIFI2, a sample of the seed of said inbred corn plant 29MMIFI2having been deposited under ATCC Accession No. 209896, wherein seed isallowed to form.
 23. The process of claim 22, further defined as aprocess of preparing hybrid corn seed, comprising crossing a firstinbred corn plant with a second, distinct inbred corn plant, whereinsaid first or second inbred corn plant is the inbred corn plant 29MIFI2,a sample of the seed of said inbred corn plant 29MIFI2 having beendeposited under ATCC Accession No.
 209896. 24. The process of claim 23,wherein crossing comprises the steps of: (a) planting in pollinatingproximity seeds of said first and second inbred corn plants; (b)cultivating the seeds of said first and second inbred corn plants intoplants that bear flowers; (c) emasculating the male flowers of saidfirst or second inbred corn plant to produce an emasculated corn plant;(d) allowing cross-pollination to occur between said first and secondinbred corn plants; and (e) harvesting seeds produced on saidemasculated corn plant.
 25. The process of claim 24, further comprisinggrowing said harvested seed to produce a hybrid corn plant.
 26. Hybridcorn seed produced by the process of claim
 23. 27. A hybrid corn plantproduced by the process of claim
 25. 28. The hybrid corn plant of claim27, wherein the plant is a first generation (F₁) hybrid corn plant. 29.The corn plant of claim 8, further comprising a single gene conversion.30. The corn plant of claim 29 wherein the single gene was stablyinserted into corn by a transgenic process.
 31. The single geneconversion of the corn plant of claim 29, where the gene is a dominantallele.
 32. The single gene conversion of the corn plant of claim 29,where the gene is a recessive allele.
 33. The single gene conversioncorn plant of claim 29, where the gene confers herbicide resistance. 34.The single gene conversion of the corn plant of claim 29, where the geneconfers insect resistance.
 35. The single gene conversion of the cornplant of claim 29, where the gene confers resistance to bacterial,fungal, or viral disease.
 36. The single gene conversion of the cornplant of claim 29, where the gene confers male fertility.
 37. The singlegene conversion of the corn plant of claim 29, where the gene conferswaxy starch.
 38. The single gene conversion of the corn plant of claim29, where the gene confers improved nutritional quality.
 39. The singlegene conversion of the corn plant of claim 29, where the gene confersenhanced yield stability.